Necrosis and hypoxia of primary and metastatic tumors have been strongly correlated with tumor aggressiveness and poor prognosis in cancer patients. Solid tumors that reach a certain size, out grow their oxygen supply and become hypoxic and eventually necrotic. In tumor areas positioned more than 70 μm from nutritive blood vessel the interstitial oxygen pressure decreases and past a distance of 150-180 μm the cells become nearly anoxic (Vaupel et al. 2001). It is believed that necrosis is the result of chronic ischemia that is caused by vascular collapse and rapid tumor cell growth that is higher than the rate of angiogenesis (Leek et al. 1999).
Necrotic areas in solid tumors undergo morphological modifications. At the beginning the original structure is basically preserved, and necrotic cells keep their overall shape but become highly eosinophilic. After some time, this pattern is replaced by liquefaction necrosis, in which the cellular structures are broken down (Leek et al. 1999).
Both necrosis and hypoxia are well established as indicators for poor prognosis. In transitional cell carcinoma of the upper urinary tract, malignant mesothelioma and renal cell carcinoma (RCC), necrosis was suggested as an independent predictor of the cancer outcome and as a very powerful tool for prognostic purposes (Edwards et al. 2003; Sengupta et al. 2005; Lee et al. 2007).
In invasive carcinoma of the breast, necrosis was correlated with high vascular density and angiogenesis, high levels of focal macrophage infiltration and decreased patient survival (Kato et al. 1997; Lee et al. 1997; Leek et al. 1999; Tomes et al. 2003). Central necrosis, which is a common feature of invasive breast cancer, was associated with poor outcome and tumor aggression. Macrophages were shown to be attracted to the necrotic tumors by chemotactic factors, released by hypoxic or dying tumor cells (Leek et al. 1999). Large necrotic areas in the ductal lumen were observed in the comedo (invasive) ductal carcinoma in situ (DCIS) as opposed to the non-comedo (non-invasive) DCIS (Cutuli et al. 2002). Necrosis and hypoxia at the center of DCIS lesions with up to 360 μm diameter, showed a marked biological difference in the nature and behavior of the neoplastic cells. Thus the presence or absence of necrosis in ducts was found to be a feasible criterion for DCIS classification (Bussolati et al. 2000).
Necrosis in the majority of this type of tumors was shown to associate with hypoxia (Tomes et al. 2003). Hypoxia and anoxia subject the tumor cells to oxidative stress. Prolonged hypoxic conditions were shown to increase the rate of mutations, to accelerate the progression of the tumor, to increase angiogenesis and metastatic potential and to activate growth promoting signaling pathways. Adaptation to oxidative stress often makes the tumor cells resistant to certain therapeutic modalities (Brown et al., 2001).
The correlation between necrosis and hypoxia is very well established, however there might be hypoxic conditions that have not reached necrosis, or necrosis that does not necessarily reflects acute or severe hypoxia (Dewhirst 1998). There are several marker genes for hypoxia, among them: hypoxia induced factor 1 (HIF1), glucose transporter 1 and carbonic anhydrase IX. Only detection of all three markers assures the classification of necrosis (Tomes et al. 2003), making the identification of an area as necrotic by gene expression quite complicated.
Necrotic and hypoxic conditions are known to create a major problem in cancer therapy. Hypoxic tumor domains are relatively resistant to radiation treatment since there is a poor promotion of the radiation assault and since stem cells that may eventually be present in the tumor volume do not respond well to the treatment, resulting in tumor re-growth (Brown et al., 1998; Dean et al., 2005). Since most chemotherapeutic reagents impose cell death due to interactions with cycling cells, cell arrest because of hypoxia results in resistance to conventional chemotherapy, leaving non-proliferating or slow proliferating cells unharmed (Tannock, 1978). Furthermore, hypoxic conditions usually create an acidic environment that might change the nature of the drug, making it less active (Tannock et al., 1989).
One of the more problematic aspects of solid tumors chemotherapy involves the trafficking of therapeutic agents into the tumors and especially to hypoxic and necrotic domains. Tumors usually contain irregular and leaky microvessels with heterogeneous blood flow and large intervessel distances. These features, in addition to the absence of proper lymphatic drainage and high interstitial pressures, make diffusion the most important mechanism of extravascular transport of nutrients and drugs in tumors. However, because of the non-regular vascularisation, many of the tumor cells are at higher distances from capillaries than cells in the normal tissues, reflected in having insufficient concentrations of antitumor agents at the cell sites. Moreover, the enhanced interstitial fluid pressure due to the lack of lymphatic drainage reduces the convection uptake and further inhibits the distribution of drugs into the tumor cells, particularly that of macromolecules (Minchinton et al., 2006).
Thus, the ability to detect hypoxic and necrotic areas within tumors in-vivo is of utmost importance. Knowledge of hypoxic tumor domains might help choosing the right treatment—either by improving tumor oxygenation before or during treatment or by using strategies that exploit the hypoxia (Weinmann et al., 2004). Using this approach, application of hypoxia-activated cytotoxins such as 2-cyclopropyl-indoloquinones, AQ4N, Tirapazamine (TPZ) and PR-104 may help improve the treatment outcome (Brown et al., 2004; Lee et al., 2007; Patterson et al. 2007).
Histopathology and immunohistochemistry are commonly used for identification of necrosis and hypoxia; however, they are invasive and do not enable detecting in situ. In situ methods include magnetic resonance imaging (MRI) (Kamel et al. 2003; Metz et al. 2003), blood oxygenation level dependent-MRI (Kennan et al. 1997), positron emission tomography (PET) (Lehtio et al. 2004) and diffusion-weighted MRI (Lang et al. 1998).
Necrosis-avid contrast agents (NACAs) for MRI can be classified into porphyrin-based and non-porphyrin-based agents. One of the most known porphyrin-based NACAs is gadophrin-2 that shows specific necrosis accumulation mostly at the margins of the necrotic area. The mechanism of accumulation was suggested to be based on serum albumin (SA) trafficking, but recent studies doubted this approach (Hofmann et al. 1999; Ni et al. 2005)
Most malignant cells cannot grow to a clinically detectable tumor mass in the absence of blood vessels. That is why tumors reaching a certain size (approximately 2-3 mm3) have to switch to an angiogenic phenotype to support their growth. The switch to an angiogenic phenotype may represent an imbalanced expression of angiogenic factors and angiogenesis inhibitors. Overexpression of angiogenic factors and down-regulation of angiogenesis inhibitors are both necessary and sufficient to induce new blood vessels growth, and these two processes usually occur simultaneously to switch on tumor angiogenesis (Cao 2005).
The biochemical features that signify blood vessels in tumors may include angiogenesis-related molecules such as certain integrins. The integrin family of cell-adhesion receptors comprises distinct 24 αβ heterodimers that recognize glycoprotein ligands in the extracellular matrix or on cell surfaces. Many members of the integrin family, including α5β1, α8β1, αIIbβ3, αVβ3, αVβ5, αVβ6 and αVβ8, recognize an Arg-Gly-Asp (RGD) motif within their ligands. These ligands include fibronectin, fibrinogen, vitronectin, von Willebrand factor and many other large glycoproteins (Takagi 2004). Hence, molecules containing RGD motif have been suggested to provide new opportunities for selective up-take and subsequently imaging and detection of primary tumor lesions, necrotic areas and targeted therapies. This field of research is getting increased attention. There are many reports of using RGD-labeled components for imaging (Temming et al. 2005). The major drawback reported in the literature is the insufficient concentration of the reporting element at the site of tumors under 4-5 mm. That is why the use of RGD-targeted imaging was mainly restricted to PET-scan, which is a more sensitive method.
Understanding tumor growth, metastases formation, tumor-host interaction and angiogenesis requires tumor models that allow easy tracking of tumor cells even at their individual level. Previous methods used for the direct measurement of most meaningful biological parameters of tumors have only been achievable via invasive end-point procedures (Lyons 2005). The majority of such methods involves histopathological examination or immunohistochemistry which are slow, invasive and not always sensitive approaches (Yang et al. 2000). Therefore, it was necessary to introduce new methods that enable direct visualization of tumor tissues, are non-invasive and enable measurement of tumor relevant parameters at both the cellular and molecular level.
In recent years several non-invasive methods have been developed: MRI and spectroscopy, PET, single photon emission computed tomography and computed tomography (Lyons 2005).
There are several imaging methods that are transgene-based. These methods enable the non-invasive measurement of a wide range of biological parameters with excellent tumor specificity, whole body imaging in live model animals and detection of metastases. Two of these methods are: bioluminescence imaging and fluorescence imaging.
Optical bioluminescence is based on three components: the enzyme luciferase, the substrate luciferin and adenosinetriphosphate (ATP). In this method, no light excitation is required to generate light emission. However, if one of these components is absent no detection is possible. The method enables monitoring cell viability or cell function at a high throughput because of the good signal/noise value (Lyons 2005). The main disadvantages of the luciferase/luciferin method are the low anatomic and image resolutions thus requiring a substantial amount of time to collect sufficient photons to form an image from an anesthetized animal. Moreover, increased tissue depth and the need for exogenous delivery of the substrate attenuate the in-vivo light emission (Yang et al., 2000; Lyons, 2005). Additionally, ex-vivo experiments are difficult to perform since ATP is required for the enzyme activity. Importantly, the method involves subjective parameterization that reduces its quantitative value.
Another way for monitoring tumor progression by optical fluorescence imaging is based on transfecting tumor cells with a stable fluorescent protein such as green fluorescent protein (GFP) and red fluorescent protein (RFP). In this method there is need for external excitation before emission can be detected. The main disadvantages of this method are that (1) the excitation and emission lights are prone to attenuation with increased tissue depth and (2) the autofluorescence of non labeled cells increases noise (Lyons, 2005). The main advantages include: multiple reporter wavelengths enabling multiplex imaging; high compatibility with a range of ex-vivo approaches for analytic methods such as fresh tissue analysis; there is no need for preparative procedures for imaging which makes it uniquely suited for visualizing in live tissue; the method is external and noninvasive; the method provides a real-time fluorescence optical imaging of internally growing tumors and metastases in transplanted animals that can give a whole-body image but also the image of single cells extracted from the primary lesion and metastases (Yang et al., 2000; Lyons, 2005). Whole body imaging is one of the most required tools for understanding tumor development. Thus, by genetically labeling of tumor cells with GFP or RFP, external whole body imaging of primary and metastatic tumors can be achieved (Yang et al. 2000).
Fluorescence tagging is suitable for in-vivo, fresh tissue and in-vitro detection. Using tumor cells expressing fluorescent proteins enables the imaging of live animals and the follow up of tumor progression in different time points. The RFP has a longer wavelength emission than GFP thus enabling higher sensitivity and resolution of microscopic tumor growth (GFP excitation wavelength—489 nm, emission wave-length—508 nm, RFP excitation wavelength—558 nm, emission wave-length—583 nm).
Ductal carcinoma in situ (DCIS) comprises a clonal proliferation of cells that appear malignant and accumulate within the lumen of the mammary ducts with no evidence of invasion into the adjacent breast stroma and beyond the epithelial basement membrane. There is a significant chance of transforming non-invasive DCIS lesions into an invasive, life-threatening disease if it is not treated at an early stage. Following the wide-spreading use of mammography, there has been a dramatic increase in the number of patients diagnosed with DCIS at the early stage and the recommended treatment modality has accordingly shifted from mastectomy (with close to 100% cure rate) toward breast conserving (BC) surgery (BCS), e.g. lumpectomy or minimally invasive breast surgery (Kepple et al., 2004), optionally accompanied by RT and adjuvant endocrine therapies. However, recurrence rates following BCS, both ipsilaterally (same breast) or contralaterally (other breast), even when accompanied by RT, were recently found to be significantly higher than after mastectomy, particularly for patients at the age of ≦40 (regression rate of 25-35%; Bijker N et al., 2006; Cutuli et al., 2002) Furthermore, multifocal lesions pose a difficulty for partial dissection and the same is true for persistently involved margins that were found critical to complete tumor regression (Cellini et al., 2005). Additionally, the physical and psychological burden and the possible cosmetic outcomes of lumpectomy followed by RT are significant. These drawbacks make the treatment and management of DCIS today controversial issues in breast cancer therapy and have stimulated the search for new and/or complementary modalities of treatment and prognosis.
DCIS is a biologically heterogeneous form of malignancy with a diverse clinical presentation, histology, cellular features, and biological potential. It has been classified into comedo (invasive) and non-comedo (non-invasive) carcinomas, where comedo has the higher grade, with a potentially more invasive subtype, characteristically containing a large necrotic area in the ductal lumen and cells with marked cytologic atypia. About two-thirds of the patients with low to intermediate grade DCIS are expected to have a multifocal, ipsilateral disease with gaps that may reach 1 cm between different foci (Cutuli et al., 2002). High-grade lesions tend to be continuous with gaps smaller than 5 mm (Cellini et al., 2005).
The natural development of non-invasive DCIS into an invasive breast tumor may take 15-20 years and involve 14 to 60 percent of the diagnosed women (Burstein et al., 2004). In fact, DCIS appears to represent a stage of breast cancer development in which many of the molecular events that define invasive breast cancer are already present (Cutuli et al., 2002; Holland et al., 1990). Specifically, ˜30% of low-grade lesions will develop into invasive carcinoma if left untreated (Sanders et al., 2005). Lesions with a diameter greater than 2.5 cm are frequently accompanied by occult microinvasive tumors that may not exceed 0.1 mm. The involvement of tumor margins provides an important prognostic marker. Close to excision (less than 1 mm) or positive margins, high-grade and/or comedo necrotic areas are associated with greater risk for recurrence.
Like in many other cancers, new blood vessel formation (angiogenesis) in breast cancer plays a central role in both local tumor progression and the development of distant metastasis (Boehm-Viswanathan, 2000; Kieran et al., 2003). Significantly higher microvessel density (MVD) was found in the DCIS tissue compared with the surrounding normal tissue (Guidi et al., 1994; Guidi et al., 1997; Guinebretière et al., 1994). Fibrocystic lesions with the highest vascular density are associated with a greater risk of breast cancer (Guidi et al., 1994; Guidi et al., 1997; Guinebretière et al., 1994). Histopathological examinations of aggressive DCIS lesions were associated with increased MVD and vascular endothelial growth factor (VEGF) expression (Guidi et al., 1997; Schneider et al., 2005). Clinicopathologic correlations also confirm the cardinal role of angiogenesis in the progression of breast cancer, making it attractive target for DCIS therapy and prognosis (Folkman, 1997; Krippl et al., 2003; Relf et al., 1997). Vessel cooption, growth by intussusception (Patan et al., 1996), vascular mimicry and vasculogenesis are naturally occurring processes that may decrease the tumor's dependence on classical angiogenesis. Of particular importance is the finding that inflammatory breast cancer depends almost entirely on vasculogenesis, apparently because of the inability of the cancer cells to bind endothelial cells.
The critical dependence of DCIS on a highly dense vascular bed has made antiangiogenic (inhibiting the formation of new blood vessels) and antivascular (occlusion/destruction of existing blood vessels) therapies (Shimizu et al., 2005; Thorpe, 2004) attractive options for localized BC therapy (Schneider et al., 2005; Folkman, 1996). Indeed, antiangiogenic drugs such as bevacizumab (an anti-VEGF-A receptor antibody) and SU011248 (an inhibitor of VEGF receptor tyrosine) are in phase II clinical trials. Interestingly, tamoxifen was also found to possess antiangiogenic activity. Yet, a growing body of evidence indicates deficiencies in the antiangiogenic approach. These include the need for a chronic treatment, the partial failure of the “resistance to resistance theory” (Schneider et al., 2005; Streubel et al., 2004) and pharmacokinetic resistance. Following these hurdles, the antivascular approach presently appears more promising, expected to result in eradication of the entire tumor with no need for chronic treatment (Folkman, 2004) A recently emerging, promising avenue for vascular-targeted treatment is by photodynamic therapy (VTP).
Likewise, targeting paramagnetic metals with appropriate relaxivity, positron emitting chemical entities (e.g. 64Cu), or fluorescence probes to the dense vascular bed of DCIS, should open new avenues for the detection of the related lesions, margins definition and prognosis as explained below. Fluorescence detection of breast cancer lesions was shown useful for up to 10 mm depth (Britton, 2006). Dynamic MRI with Gd as a contrast agent is based on enhanced leakiness of the tumor vasculature and currently used for tumor localization in the breast (Rankin, 2000). However, the current use of MRI is limited by the available short integration time of contrast agents that shortly reside but do not selectively taken up by the tumor tissue.
Photodynamic therapy (PDT) generates a burst of cytotoxic reactive oxygen species (ROS) at a selected treatment site. Because of their short lifetime, the ROS toxicity is confined to the illuminated site. PDT typically consists of five steps: 1. Intravenously (IV) administration of a photosensitizer; 2. A time period that enables a desirable concentration of photosensitizers to reach the target tissue; 3. Illumination of the target tissue transcutaneously or interstitially with high intensity laser light (up to 1 W for continuous illumination) via thin (0.4 mm diameter or less) optical fibers for deep tissue illumination with the consequent local generation of cytotoxic ROS; 4. Development of tumor necrosis and tumor eradication; 5. Tissue remodeling and healing.
Vascular-targeted PDT (VTP) aims at ROS generation within the blood vessels of the treated tissue that can be accomplished either by tissue illumination immediately after sensitizer's administration or by using sensitizers that do not extravasate from the circulation. Several generations of bacteriochlorophyll sensitizers termed herein “Bchl derivatives” or “BchlD” have been developed in our laboratory. The synthesized compounds (Rosenbach-Belkin et al., 1996; U.S. Pat. No. 5,650,292) possess a very strong absorption in the NIR (750-765 nm) enabling deep light penetration into the subject tissues, assuring a treatment diameter of up to 4 cm around a cylindrical fiber at high fluence rates (20 mW-1 W). Upon illumination, a local high concentration of ROS (OH. and O2− radicals) is generated in the tumor and the vicinity by the circulating BchlD, resulting in blood clotting and tumor vessels perforation followed by a complete arrest of the tumor vasculature within minutes of illumination. With some Bchl derivatives, direct intoxication of the endothelial cells was observed (Gross et al., 2003; Mazor et al., 2005). For reasons that are presently under investigation, the tumor vascular response is markedly faster and harsher compared with that of the vessels in the surrounding normal tissue. Treatment efficacy results in high cure rates (60-90% animal survival) (Mazor et al., 2005). Importantly, the IV injected sensitizers clears rapidly from the circulation of the treated animals (T1/2 is in the order of minutes to hours, (Mazor et al., 2005) avoiding prolonged skin toxicity and allowing for treatment repetition if needed. In Phase II clinical trials on localized prostate cancer in patients, where radiation therapy failed (Weersink et al., 2005), BchlD-based VTP has generally resulted in a successful eradication of the tumor lesions at 50-60% of the treated patients and remodeling of the tissue. A second treatment in both animal models and humans (phase II/III clinical trials) appear to result in similar or higher cure rates per session (depending on the drug and light dose), increasing the expected overall rate of success to ˜90% after 2-3 sessions. Importantly, markedly higher cure rates per session were found in animal studies with higher doses of the applied sensitizer.
Photodynamic therapy (PDT) in tumors involves the combination of administered photosensitizer and local light delivery, both innocuous agents by themselves, but in the presence of molecular oxygen they are capable of producing cytotoxic reactive oxygen species (ROS) that can eliminate cells. Being a binary treatment modality, PDT allows for greater specificity, and has the potential of being more selective yet not less destructive when compared with commonly used chemotherapy or radiotherapy (Dougherty et al. 1998).
Application of novel bacteriochlorophyll (Bchl) derivatives as sensitizers in PDT has been reported by the present inventors in recent years (Zilberstein et al., 2001; Schreiber et al., 2002; Gross et al., 1997; Zilberstein et al., 1997; Rosenbach-Belkin et al., 1996; Gross et al., 2003a; Koudinova et al., 2003; Preise et al., 2003; Gross et al., 2003b) and in the patent publications U.S. Pat. Nos. 5,726,169 5,650,292, 5,955,585, 6,147,195, 6,740,637, 6,333,319, 6,569,846, 7,045,117, DE 41 21 876, EP 1 246 826, WO 2004/045492, WO 2005/120573. The spectra, photophysics, and photochemistry of Bchl derivatives have made them optimal light-harvesting molecules with clear advantages over other sensitizers presently used in PDT. These Bchl derivatives are mostly polar and remain in the circulation for a very short time with practically no extravasation into other tissues (Brandis, 2003; Mazor et al. 2005). Therefore, these compounds are good candidates for vascular targeted PDT (VTP) that relies on short (5-10 min) temporal intravascular encounter with light and higher susceptibility of the tumor vessels to the PDT-generated cytotoxic ROS.
Recent studies performed by our group showed that primary photosensitization is intravascular with rapid development of ischemic occlusions and stasis within the illumination period. This process also promotes photochemically induced lipid peroxidation (LPO) and early endothelial cell death that is primarily confined to the tumor vasculature (Koudinova et al. 2003). Due to light independent progression of free radical chain reactions along with developing hypoxia, LPO and cell death spread beyond the vascular compartment to cover the entire tumor interstitium until complete necrosis of the tumor is attained around 24 hours post PDT. Hence, the primary action of PDT blocks blood supply and induces hypoxia that initiates, in a secondary manner, a series of molecular and patho-physiological events that culminate with tumor eradication. Importantly, this approach relies on the differences between the response of normal and tumor blood vessels to the generated ROS.
International Application No. WO 2008/023378 of the same applicants, hereby incorporated by reference in its entirety as if fully disclosed herein, discloses novel conjugates of porphyrin, chlorophyll and bacteriochlorophyll (Bchl) derivatives with peptides containing the RGD motif or with RGD peptidomimetics, and their use in methods of in-vivo photodynamic therapy and diagnosis of tumors and different vascular diseases such as age-related macular degeneration. In particular, the Bchl derivative c(RGDfK)-Pd-MLT (Compound 24) showed accumulation of up to 4-8 μM in xenografts of primary tumors and stays at the tumor site for prolonged time enabling accumulation of the signal and a good signal to noise ratio.
Fluorescence tagging is suitable for in vivo, fresh tissue and in vitro detection. c(RGDfK)-Pd-MLT has intrinsic fluorescence in the near infra red (NIR) that can be detected. c(RGDfK)-2H-MLT has three orders of magnitude higher glowing ability and therefore might be an even better candidate for targeted imaging. In this study we showed that these molecules open the possibility to accurately detect tumor margins and necrosis in human breast adenocarcinoma model. Detecting tumor margins and necrosis present up to-date, two of the most challenging issues in tumor treatment. Moreover, both are faithful predictors of tumor re-growth after treatment. Thus, in the future, when clinically applied, the aforementioned RGD derivatives are expected to be suitable for tumor and necrosis detection on the operating table.